Determination of 2,3-dinor-6-ketoprostaglandin F1 alpha in urine samples by liquid chromatography and radioimmunoassay

A method for 2,3-dinor-6-ketoprostaglandin F1 alpha quantification based on high-performance liquid chromatography-radioimmunoassay is described. Samples are acidified to pH 3 and processed through C18 disposable cartridges. The prostanoids are eluted with methyl formate and further separated on a reversed-phase column using acetonitrile-acetic acid-triethylamine buffer (32:68). Studies of the effect of eluent pH were performed in order to optimize resolution and separation of 2,3-dinor-6-keto-PGF1 alpha from other prostanoids. Eluates were collected and assayed by radioimmunoassay using a heterologous system, with 6-keto-PGF1 alpha as radioligand and an antiserum with high cross-reactivity for 2,3-dinor-6-keto-PGF1 alpha. Sensitivity, precision and accuracy of the assay procedure are reported together with the validation of its specificity. The proposed method has been applied to the determination of this prostacyclin metabolite in human urine.     

Solid- and liquid-phase extraction for the gas chromatographic-tandem mass spectrometric quantification of 2,3-dinor-thromboxane B2 and 2,3-dinor-6-oxo-prostaglandin F1 alpha in human urine

Whole body synthesis of thromboxane A2 is best assessed by quantifying non-invasively its major urinary metabolite, i.e., 2,3-dinor-thromboxane B2 (2,3-dn-TxB2), by gas chromatography-mass spectrometry (GC-MS) or GC-tandem MS. Methods based on these techniques usually require a series of extraction and purification procedures including solid-phase extraction (SPE) and thin-layer chromatography (TLC) or liquid chromatographic separation of authentic or derivatized 2,3-dn-TxB2. Taking advantage of the inherent accuracy of GC-tandem MS and the high selectivity of the extraction of methoximated 2,3-dn-TxB2 on phenylboronic acid SPE cartridges we developed a method that involves only SPE steps prior to quantification by GC-tandem MS. The method was validated by performing in parallel an additional TLC step. Method mean accuracy and precision were of the order of 103% and 95%, respectively. The method allows furthermore co-processing of the same urine sample to quantify accurately and rapidly the major urinary metabolite of prostacyclin, i.e., 2,3-dn-6-oxo-prostaglandin (PG) F1 alpha, by GC-tandem MS. The limit of detection of the method was below each 5 pg of 2,3-dn-TxB2 and 2,3-dn-6-oxo-PGF1 alpha per 5 ml of urine. Our study suggests that dinor metabolites of isothromboxanes and isoprostacyclins are not abundantly present in human urine.     

Assay methods for 6-keto-prostaglandin F1 alpha in human urine. Comparison of chromatographic techniques with radioimmunoassay and gas chromatography-negative-ion chemical-ionization mass spectrometry

6-Keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) in human urine is considered to be a reflection of renal prostacyclin production. Because of the large amounts of unidentified eicosanoid metabolites in urine that may potentially bind to 6-keto-PGF1 alpha antisera, most radioimmunoassays include chromatographic purification of urine. A comparison of chromatographic techniques and of antisera to 6-keto-PGF1 alpha for the assay of human urine is described. Gas chromatography--negative-ion chemical-ionization mass spectrometry (GC--NICI-MS) was used as the reference method. Radioimmunoassays were performed with each of four antisera combined with each of three chromatographic purification systems (silicic acid, Sephadex LH-20, reversed-phase high-performance liquid chromatography). There was marked variability in the results; however, there was at least one chromatographic method for each antiserum that yielded results comparable to GC--NICI-MS. Direct radioimmunoassay of urine without chromatography yielded markedly elevated and variable results for the four antisera. In contrast, the four antisera gave very similar results with direct assay of media from isolated perfused organs. Thus, for the radioimmunoassay of 6-keto-PGF1 alpha in human urine, each antiserum is sensitive to different contaminants in urine and must be individually matched to a chromatographic purification system.     

Diethylhydrogensilyl-cyclic diethylsilylene derivatives in gas chromatography-mass spectrometry of prostanoids. III. F alpha-prostaglandins and thromboxane B2 derivatives

The gas chromatographic (GC) and GC-mass spectrometric properties of the diethylhydrogensilyl-cyclic diethylsilylene (DEHS-DES) derivatives of prostaglandin (PG) F1 alpha methyl ester, PGF2 alpha methyl ester, 6-keto-PGF1 alpha methyl ester-alkyloxime and thromboxane (TX) B2 methyl ester-alkyloxime and the DES derivative of 13,14-dihydro-15-keto-PGF2 alpha methyl ester-alkyloxime were studied. When the ketonic PGs and TXB2 were converted into their methyloxime derivatives, the methylene unit values of these five prostanoid derivatives were slightly greater than those of the corresponding dimethylethylsilyl ether derivatives. When the ketonic PGs were converted into their corresponding ethyloxime derivatives, baseline separation was achieved in 20 min by use of a methylsilicone cross-linked fused-silica capillary column. The mass spectra of these derivatives were characterized by the ion at m/z 157 for F alpha prostaglandins and m/z 269 for TXB2. The major fragmentations were directed by the DES group, and other fragmentations common to the prostanoid derivatives were losses of an ethyl radical at the silicon atom, C5H11 hydrocarbon fragment, diethylhydrogensilanol and C15-C20 hydrocarbon fragment. The mass fragmentations of these prostanoid derivatives are briefly discussed. GC with high-resolution selected-ion monitoring was carried out for the TXB2 derivative at a resolution of 8000 by monitoring the ion at m/z 269.1573. A 25-pg amount of this derivative showed a well shaped doublet with a signal-to-noise ratio of more than 300:1.     

Synthesis of 6-hydroxylated bile acids and identification of 3 alpha,6 alpha,7 alpha,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acid in human meconium and neonatal urine

Three 6-hydroxylated bile acids, 3 alpha,6 alpha,7 alpha,12 alpha-, 3 alpha,6 beta,7 alpha,12 alpha- and 3 alpha,6 beta,7 beta,12 alpha-tetrahydroxy-5 beta-cholan-24-oic acids, were synthesized from methyl cholate, and a sensitive method was developed for analyzing them by gas chromatography-mass spectrometry for the stoichiometric study of fetal bile acids. 3 alpha,6 alpha,7 alpha,12 alpha-Tetrahydroxy-5 beta-cholan-24-oic acid (6 alpha-hydroxylated cholic acid) was identified from human meconium and healthy neonatal urine by comparison with the mass spectrum of the reference compound. In human meconium, 6 alpha-hydroxylated cholic and chenodeoxycholic acids were determined in 1.2% and 29.0% of the total bile acids, respectively. We discuss the significance of hydroxylation at the C-1 beta and C-6 alpha positions of bile acids and their elimination in fetal and neonatal periods.     

Effect of magnesium lithospermate B on urinary excretion of arachidonate metabolites in rats with renal failure

The effect of magnesium lithospermate B isolated from Salviae miltiorrhizae Radix on excretion of urinary arachidonate metabolites was examined in both normal rats and those given adenine. Urinary excretion of prostaglandin E2 (PGE2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) decreased while urinary thromboxane B2 (TXB2) excretion increased markedly with the progression of renal failure. Rats administered magnesium lithospermate B showed an increase of urinary PGE2 excretion at the 6th and 12th days. Excretion of 6-keto-PGF1 alpha also showed a significant increase on the 6th and 12th days in rats with renal failure induced by the administration of adenine. However, these effects were lower than the corresponding values in normal rats. In addition, urinary PGE2 and 6-keto-PGF1 alpha excretions showed no appreciable difference in rats that exhibited progressive renal failure with continuation of the adenine administration period, as shown on the 18th and 24th days. There were no significant changes in TXB2 excretion between the control and magnesium lithospermate B-treated groups throughout the experimental period.     

Setting up a 2D-LC/MS/MS method for the rapid quantitation of the prostanoid metabolites 6-oxo-PGF(1alpha) and TXB2 as markers for hemostasis assessment

6-oxo-PGF(1alpha) and TXB(2) are the metabolites of the prostanglandin PGI(2) and of the thromboxane TXA(2), respectively. PGI(2) and TXA(2) are arachidonic acid-derived compounds which regulate the blood hemostasis. Their quick metabolism leads to the 6-oxo-PGF(1alpha) and TXB(2) metabolites in plasma. In order to study on a large base the external factors influencing the hemostatic conditions, there is a need for a fast and reliable assay for quantitating these metabolites. Some methods have been published for the analysis of the arachidonic acid-derived compounds and some are dealing with mass spectrometry but nonspecifically centered on these specific compounds with a fast and cheap protocol, amenable for large-scale studies. Here we describe an analytical strategy that incorporates a two-dimensional chromatography running coupled to tandem mass spectrometry that minimizes the sample preparation and addresses the presence of the TXB(2) anomers for a robust quantitation measurement. After a protein precipitation, 100 microl of the supernatant (corresponding to 50 microl of the original plasma) was injected in a two-dimensional chromatographic system which operates an on-line clean-up and a subsequent chromatographic separation of the targeted analytes with a limit of quantitation (LOQ) of 22 pg/ml for 6-oxo-PGF(1alpha), and and a LOQ of 25 pg/ml for TXB(2).     

Solid-phase spectrophotometric determination of trace amounts of vanadium using 2,3-dichloro-6(3-carboxy-2- hydroxynaphthylazo)quinoxaline

Solid-phase spectrophotometry (SPS) has been applied to analysis for trace amounts of vanadium in several environmental water (potable and polluted), biological samples (human blood and urine), and soil samples. Vanadium was sorbed in a styrene-divinylbenzene-type anion-exchanger Dowex 1-X8 as a vanadium--2,3-dichloro-6(3-carboxy-2-hydroxynaphthylazo)quinoxaline. Resin phase absorbances at 606 and 800 nm were measured directly which allowed the determination of vanadium in the range 0.03-2.2 ng ml(-1) with a relative standard deviation (R.S.D.) of 1.4%. The comparison of the SPS method and the gallic acid persulphate method shows that the linearity, analytical sensitivity, and precision were better for the SPS method, and that the latter method has lower detection and quantification limits compared with the gallic acid persulphate method.     

Simultaneous determination of prostanoids in plasma by gas chromatography-negative-ion chemical-ionization mass spectrometry

A method for simultaneous determination of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), and thromboxane B2 (TxB2) in plasma was developed. After acidification and addition of 2H- and 3H-labelled internal standards, plasma prostanoids were extracted by reversed-phase cartridges and purified by normal-phase high-performance liquid chromatography. The pentafluorobenzyl, methoxime, trimethylsilyl derivatives were formed. Negative-ion chemical-ionization mass spectra with methane as reagent gas show one intense peak at m/z (M - pentafluorobenzyl). This ion was used for selective-ion monitoring. Prostanoid plasma concentrations (pg/ml) in five healthy volunteers were: PGE2 2.0-10.4, PGF2 alpha 2.2-9.8, 6-keto-PGF1 alpha 0.6-1.8, and TxB2 3.0-45.3. However, there is evidence that the TxB2 values may frequently be falsely high because of ex vivo production during the sampling procedure.     

Identification of 2,3-dimethyl-2,3-diisobutyl succinonitrile in laser printer emissions

2,3-Dimethyl-2,3-diisobutyl succinonitrile was identified as the main volatile organic compound (>90%) emitted from laser printers during the printing process. Experiments were carried out in a large environmental chamber of 30 m3, where the printers were placed and working simulating 'real office setting' conditions. Air samples were taken on Tenax TA adsorbent cartridges in the vicinity of the printers and further analyzed by thermal desorption gas chromatography/mass spectrometry (TDGC/MS). The structure of the compound has been determined and is presented in this study. Additional data obtained by nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) spectroscopy, and liquid chromatography/tandem mass spectrometry (LC/MS/MS) support the proposed structure, with no reported CAS number, as 2,3-dimethyl-2,3-diisobutyl succinonitrile. It is a byproduct of the thermal decomposition of 2,2'-azobis(2,4-dimethyl valeronitrile), a commercially available free radical polymerization initiator used in polymerization processes during the manufacture of the toners. By means of head-space GC/MS, 15 toners used in black & white and colour printers have been investigated. Six of them contained 2,3-dimethyl-2,3-diisobutyl succinonitrile, which has also been detected in the respective processed paper.     

Simultaneous analysis of prostanoids using liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry

A liquid chromatography/high-field asymmetric waveform ion mobility spectrometry/tandem mass spectrometry (LC-FAIMS-MS/MS) semi-quantitative method was developed for the simultaneous determination of prostaglandin (PG) E2, PGD2, PGF(2alpha), 6-keto-PGF(1alpha), and thromboxane (TX) B2. Diluted samples containing these prostanoids and their tetra-deuterium-substituted internal standards were analyzed by LC followed by either selected reaction monitoring (LC-SRM) or FAIMS and selected reaction monitoring (LC-FRM). FAIMS reduced background noise, separated the isobaric ions PGE2 and PGD2, and separated dynamically interchanging TXB2 anomers. This is the first report of the separation of interconverting anomers by FAIMS. Generally, the LC-FRM chromatograms were more selective than the LC-SRM chromatograms. Its potential was demonstrated by analysis of prostanoids in guinea pig lumbar spinal cord homogenate.     

Profiling of eicosanoids in inflamed gall bladder wall by gas chromatography with selected-ion monitoring.

The profiling of eicosanoids, including prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), thromboxane B2 (TXB2) and leukotriene B4 (LTB4), in dog and human gall bladders was carried out by a combination of an effective and convenient clean-up procedure and gas chromatography with selected-ion monitoring. The clean-up procedure was based on the stepwise elution of their methyl ester derivatives from a silica gel column with n-hexane-ethyl acetate and ethyl acetate-methanol in various ratios. The LTB4 methyl ester was eluted with an n-hexane-ethyl acetate (2:1, v/v) fraction because LTB4 is more lipophilic than the other eicosanoids. The present method permitted the quantitation of trace amounts of eicosanoids, including LTB4, present in tissues in the order of pg/mg of protein, without interference from other endogenous substances. In experimental acalculous cholecystitis produced in dog, the levels of eicosanoids (except LTB4) were significantly changed. Of these eicosanoids, the level of 6-keto-PGF1 alpha was significantly higher in the seromuscular layer and correlated with the observed severe morphological changes. In human chronic cholecystitis with gallstones, the level of 6-keto-PGF1 alpha in the mucosal layer was significantly higher than that in the seromuscular layer. These data suggest that prostaglandin I2 may play an important pathophysiological role in the course of cholecystitis.     

Determination of salbutamol enantiomers in human urine using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin in nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) was successfully applied to the resolution and the determination of salbutamol enantiomers in urine samples using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin (HDAS-beta-CD). After optimization of the electrophoretic parameters, namely the background electrolyte (BGE) composition and the HDAS-beta-CD concentration, salbutamol enantiomers were completely resolved using a BGE made up of 10 mM ammonium formate and 15 mM HDAS-beta-CD in methanol acidified with 0.75 M formic acid. Isoprenaline was selected as internal standard. Solid-phase extraction (SPE) was used for sample cleanup prior to the CE separation. Different sorbents involving polar, nonpolar interactions or dual retention mechanisms were evaluated and extraction cartridges containing both nonpolar and strong cation-exchange functionalities were finally selected. Salbutamol enantiomers recoveries from urine samples were determined. The method was then successfully validated using a new approach based on accuracy profiles over a concentration range from 375 to 7500 ng/mL for each enantiomer.     

2,3-di-O-methoxymethyl-6-O-tert-butyldimethylsilyl-beta-cyclodextrin, a useful stationary phase for gas chromatographic separation of enantiomers

Heptakis(2,3-di-O-methoxymethyl-6-O-tert-butyldimethylsilyl)-beta-cyclodextrin (2,3-MOM-6-TBDMS-beta-CD), synthesized by using methoxymethylchloride (MOM-Cl) as derivatization reagent, was used for capillary gas chromatographic separation of enantiomers. The new chiral stationary phase proved to be suitable for the enantiodifferentiation of volatiles from various chemical classes. Compared to the corresponding gamma-CD derivative (2,3-MOM-6-TBDMS-gamma-CD), the spectrum of compounds for which enantiomers could be separated was more limited and the enantioseparation achieved was generally less pronounced. Unusually high separation factors were observed for 2-alkyl esters of short chain acids (C2-C6). Phenomena underlying the enantioseparation of 2-pentyl acetate (alpha: 4.31; 35 degrees C) were investigated by determining thermodynamic parameters. Data show that only one enantiomer is retained significantly on the chiral stationary phase whereas the other one behaves like the hydrocarbons used as references.     

Detection of trace levels of thiodiglycol in blood, plasma and urine using gas chromatography-electron-capture negative-ion chemical ionisation mass spectrometry

A sensitive method has been developed for the detection and quantitative determination of thiodiglycol in blood, plasma and urine. Samples were extracted from Clin Elut columns and cleaned up on C18 Sep-Pak cartridges (blood, plasma) or Florisil Sep-Pak cartridges (urine). Tetradeuterothiodiglycol was added to the sample prior to extraction as internal standard. Thiodiglycol was converted to its bis-(pentafluorobenzoate) derivative and analysed by capillary gas chromatography-electron-capture negative-ion chemical ionisation mass spectrometry using selected ion monitoring. Levels of thiodiglycol down to 1 ng/ml (1 ppb) could be detected in 1-ml spiked blood and urine samples; calibration curves were linear over the range 5- or 10-100 ng/ml. Blood and urine samples from a number of control subjects were analysed for background levels of thiodiglycol. Concentrations up to 16 ng/ml were found in blood, but urine levels were below 1 ng/ml.     

High-performance liquid chromatographic detection of myocardial prostaglandins and thromboxanes

Reperfusion of ischemic myocardium is associated with the breakdown of membrane phospholipids and a corresponding increase in arachidonic acid, ultimately resulting in the production of prostaglandins (PGs) and thromboxanes (TXs). However, quantification of these arachidonic acid metabolites has been limited to radioimmunoassay because of their presence in extremely low amounts. In this report, we describe a method suitable to detect sub-picogram levels of 6-keto-PGF1 alpha, PGF1 alpha, PGE2 and TXB2 in myocardial perfusates by high-performance liquid chromatography (HPLC) with a high-gain photomultiplier and a xenon-mercury are lamp. Strong Raleigh scatter of the lamp was eliminated by both interference and long-pass cut-off filters. Improved sample clean-up and HPLC separation were achieved by an HPLC system with an Ultrasphere 3-microns C18 column.     

Evaluation of a radioimmunoassay of alpha 1-microglobulin (alpha 1-m) with simplified procedures

A newly established double antibody radioimmunoassay (RIA) was fundamentally and clinically evaluated. Original procedures were partially modified as follows: Sample volume for serum and urine was changed to 25 microliters, and thus 200 mg/l of alpha 1-m standard was prepared using 50 microliters of original standard solution (100 mg/l). The results were satisfactory in sensitivity (0.3 mg/l obtained from -2SD method), intra-assay precision with its coefficient variation (CV) ranging from 3.0 to 7.4%, interassay precision with its CV ranging from 3.0 to 10.7%, and recovery with the mean value of 102.4% in serum and 108.2% in urine respectively. There were no changes about alpha 1-m value between diluted (2 times) and undiluted with high concentration samples. Normal levels of alpha 1-m were less than 25 mg/l in serum and less than 10 mg/l in urine. The present results indicate that the determination of alpha 1-m could be very simple and useful for the most sensitive screening test for the evaluation of renal function.     

Quantitative analysis of 6-keto-prostaglandin F1 alpha using immunoaffinity purification and gas chromatography-mass spectrometry

This paper describes an immunoaffinity purification technique for 6-keto-prostaglandin F1 alpha (6KPGF1 alpha) prior to quantitative analysis by high-resolution gas chromatography-negative-ion chemical ionization mass spectrometry (HRGC-NICIMS). Polyclonal antibodies to 6KPGF1 alpha were partially purified using Staphylococcus aureus Protein A immobilized on Sepharose CL-4B. This partially purified fraction was covalently bound to silica gel using N-hydroxysuccinimidyl-functionalized silica. Columns constructed using this gel quantitatively bound 6KPGF1 alpha which could be eluted quantitatively with acetonitrile-water (19:1). Binding capacity was reconstituted by washing with 0.01 M phosphate buffer (pH 7.4). Human urinary and canine plasma 6KPGF1 alpha was sufficiently purified using these columns that HRGC-NICIMS analysis of the methoxime-pentafluorobenzyl-tris-trimethylsilyl derivative was interference-free.     

Synthesis of the core tetrasaccharide of Trypanosoma cruzi glycoinositolphospholipids: Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4

[structure: see text] Synthesis of the core tetrasaccharide Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4, found in glycoinositolphospholipids of Trypanosoma cruzi parasites, is described. The key building block, 6-O-(2-azido-3-O-benzyl-6-O-((2-benzyloxycarbonylaminoethyl)phosphonic acid benzyl ester)-2-deoxy-alpha-D-glucopyranosyl)-1-di-O-benzylphosphoryl-4,5-O-isopropylidene-2,3-O-(D-1,7,7-trimethyl[2,2,1]bicyclohept-6-ylidene)-D-myo-inositol, was synthesized using a partially protected glucosyl D-camphorinositolphosphate and a (2-benzyloxycarbonylaminoethyl)phosphonic acid derivative in a regioselective phosphonate esterfication. Elongation with ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzyl-1-alpha-D-thiomannopyranoside using dimethyl(methylthio)sulfonium trifluoromethanesulfonate gave a fully protected tetrasaccharide which was successfully deprotected subsequently with sodium methoxide, sodium in liquid ammonia, and aq hydrochloric acid to give title compound.     

Mass fragmentographic determination of prostaglandin F2 alpha in human and rabbit urine

Analysis of prostaglandin F2 alpha (PGF2 alpha) in urine is a useful indicator of renal prostaglandin synthesis. A mass fragmentographic method for PGF2 alpha analysis in human urine was developed using [3,3,4,4-2H4]PGF2 alpha as an internal standard and carrier. PGF2 alpha was extracted from urine (20 ml) with chloroform, purified by preparative thin-layer chromatography and converted to the methyl ester trimethylsilyl ether before analysis by gas chromatography--mass spectrometry. The specificity of the urine analysis was demonstrated by retention time and the use of two pairs of fragments m/e 494/498 and 513/517 with the same results. The coefficient of variation for duplicate analysis averaged 12.6%, n = 17. Urine from recumbent women contained 4.9 +/- 2.6 (S.D.) ng/ml or 4.1 +/- 1.0 ng PGF2 alpha per mg creatinine (n = 10) with little diurnal variation. Male urine contained 5.0 +/- 2.7 (S.D.) ng/ml or 3.7 +/- 2.1 ng/mg creatinine (n = 10). Similar concentrations were found in boys and in girls. These observations indicate that urinary PGF2 alpha originates from the kidneys with little contribution from the male accessory sexual glands. This method can also be applied to analysis of PGF2 alpha in rabbit urine.